Lotun, A;Li, D;Xu, H;Su, Q;Tuncer, S;Sanmiguel, J;Mooney, M;Baer, CE;Ulbrich, R;Eyles, SJ;Strittmatter, L;Hayward, LJ;Gessler, DJ;Gao, G;
PMID: 37149081 | DOI: 10.1016/j.pneurobio.2023.102460
Myelinating oligodendrocytes are essential for neuronal communication and homeostasis of the central nervous system (CNS). One of the most abundant molecules in the mammalian CNS is N-acetylaspartate (NAA), which is catabolized into L-aspartate and acetate by the enzyme aspartoacylase (ASPA) in oligodendrocytes. The resulting acetate moiety is thought to contribute to myelin lipid synthesis. In addition, affected NAA metabolism has been implicated in several neurological disorders, including leukodystrophies and demyelinating diseases such as multiple sclerosis. Genetic disruption of ASPA function causes Canavan disease, which is hallmarked by increased NAA levels, myelin and neuronal loss, large vacuole formation in the CNS, and early death in childhood. Although NAA's direct role in the CNS is inconclusive, in peripheral adipose tissue, NAA-derived acetate has been found to modify histones, a mechanism known to be involved in epigenetic regulation of cell differentiation. We hypothesize that a lack of cellular differentiation in the brain contributes to the disruption of myelination and neurodegeneration in diseases with altered NAA metabolism, such as Canavan disease. Our study demonstrates that loss of functional Aspa in mice disrupts myelination and shifts the transcriptional expression of neuronal and oligodendrocyte markers towards less differentiated stages in a spatiotemporal manner. Upon re-expression of ASPA, these oligodendrocyte and neuronal lineage markers are either improved or normalized, suggesting that NAA breakdown by Aspa plays an essential role in the maturation of neurons and oligodendrocytes. Also, this effect of ASPA re-expression is blunted in old mice, potentially due to limited ability of neuronal, rather than oligodendrocyte, recovery.
Tang N, Cheng C, Zhang X, Qiao M, Li N, Mu W, Wei XF, Han W, Wang H
PMID: 31999649 | DOI: 10.1172/jci.insight.133977
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In recent years, chimeric antigen receptor-modified T cell (CAR T cell) therapy has proven to be a promising approach against cancer. Nonetheless, this approach still faces multiple challenges in eliminating solid tumors, one of which being the immunosuppressive tumor microenvironment (TME). Here, we demonstrated that knocking out the endogenous TGF-? receptor II (TGFBR2) in CAR T cells with CRISPR/Cas9 technology could reduce the induced Treg conversion and prevent the exhaustion of CAR T ce lls. Meanwhile, TGFBR2-edited CAR T cells had better in vivo tumor elimination efficacy, both in cell line-derived xenograft and patient-derived xenograft solid tumor models, whether administered locally or systemically. In addition, the TGFBR2-edited CAR T cells could eliminate contralaterally reinoculated xenografts in mice effectively, with an increased proportion of memory subsets within circulating CAR T cells of central memory and effector memory subsets. In conclusion, we greatly improved the in vitro and in vivo function of CAR T cells in TGF-?-rich tumor environments by knocking out endogenous TGFBR2 and propose a potentially new method to improve the efficacy of CAR T cell therapy for treating solid tumors
Rigoni R, Fontana E, Dobbs K, Marrella V, Taverniti V, Maina V, Facoetti A, D'Amico G, Al-Herz W, Cruz-Munoz ME, Schuetz C, Gennery AR, Garabedian EK, Giliani S, Draper D, Dbaibo G, Geha RS, Meyts I1, Tousseyn T, Neven B, Moshous D, Fischer A, Schulz A, Finocchi A, Kuhns DB, Fink DL, Lionakis MS, Swamydas M, Guglielmetti S, Alejo J, Myles IA, Pittaluga S, Notarangelo LD, Villa A, Cassani B
PMID: 32311393 | DOI: 10.1016/j.jaci.2020.04.005
BACKGROUND:
Severe early-onset erythroderma and gut inflammation, with massive tissue infiltration of oligoclonal activated T cells are the hallmark of Omenn Syndrome (OS).
OBJECTIVE:
The impact of altered gut homeostasis in the cutaneous manifestations of OS remains to be clarified.
METHODS:
We analyzed a cohort of 15 patients with OS and the Rag2R229Q mouse model. Homing phenotype of circulating lymphocytes were analyzed by flow cytometry. Inflammatory cytokines and chemokines were examined in the sera by ELISA and in skin biopsies by immunohistochemistry and in situ RNA hybridization. Experimental colitis was induced in mice by dextran sulfate sodium salt (DSS).
RESULTS:
We show that memory/activated T cells from OS patients and from the Rag2R229Q mouse model of OS abundantly express the skin homing receptors Cutaneous Lymphocyte Associated Antigen (CLA) and CCR4, associated with high levels of CCL17 and CCL22 chemokines. Serum levels of LPS are also elevated. A broad Th1/Th2/Th17 inflammatory signature is detected in the periphery and in the skin. Increased Tlr4 expression in the skin of Rag2R229Q mice is associated with enhanced cutaneous inflammation upon local and systemic administration of LPS. Likewise, boosting colitis in Rag2R229Q mice results in increased frequency of CCR4+ splenic T cells and worsening of skin inflammation, as indicated by epidermal thickening, enhanced epithelial cell activation and dermal infiltration by Th1 effector T cells.
CONCLUSIONS:
These results support the existence of an interplay between gut and skin that can sustain skin inflammation in O
Cytokine RNA In Situ Hybridization Permits Individualized Molecular Phenotyping in Biopsies of Psoriasis and Atopic Dermatitis
Wang, A;Fogel, A;Murphy, M;Panse, G;McGeary, M;McNiff, J;Bosenberg, M;Vesely, M;Cohen, J;Ko, C;King, B;Damsky, W;
| DOI: 10.1016/j.xjidi.2021.100021
Detection of individual cytokines in routine biopsies from patients with inflammatory skin diseases has the potential to personalize diagnosis and treatment selection, but this approach has been limited by technical feasibility. We evaluate whether a chromogen-based RNA in situ hybridization approach can be used to detect druggable cytokines in psoriasis and atopic dermatitis. A series of psoriasis (n = 20) and atopic dermatitis (n = 26) biopsies were stained using RNA in situ hybridization for IL4, IL12B (IL-12/23 p40), IL13, IL17A, IL17F, IL22, IL23A (IL-23 p19), IL31, and TNF (TNF-α). NOS2 and IFNG, canonical psoriasis biomarkers, were also included. All 20 of the psoriasis cases were positive for IL17A, which tended to be the predominant cytokine, although some cases had relatively higher levels of IL12B, IL17F, or IL23A. The majority of cytokine expression in psoriasis was epidermal. A total of 22 of 26 atopic dermatitis cases were positive for IL13, also at varying levels; a subset of cases had significant IL4, IL22, or IL31 expression. Patterns were validated in independent bulk RNA-sequencing and single-cell RNA-sequencing datasets. Overall, RNA in situ hybridization for cytokines appears highly specific with virtually no background staining and may allow for individualized evaluation of treatment-relevant cytokine targets in biopsies from patients with inflammatory skin disorders.
Translatomic analysis of regenerating and degenerating spinal motor neurons in injury and ALS
Shadrach, J;Stansberry, W;Milen, A;Ives, R;Fogarty, E;Antonellis, A;Pierchala, B;
| DOI: 10.1016/j.isci.2021.102700
The neuromuscular junction is a synapse critical for muscle strength and coordinated motor function. Unlike CNS injuries, motor neurons mount robust regenerative responses after peripheral nerve injuries. Conversely, motor neurons selectively degenerate in diseases such as amyotrophic lateral sclerosis (ALS). To assess how these insults affect motor neurons in vivo, we performed ribosomal profiling of mouse motor neurons. Motor neuron-specific transcripts were isolated from spinal cords following sciatic nerve crush, a model of acute injury and regeneration, and in the SOD1G93A ALS model. Of the 267 transcripts upregulated after nerve crush, 38% were also upregulated in SOD1G93A motor neurons. However, most upregulated genes in injured and ALS motor neurons were context specific. Some of the most significantly upregulated transcripts in both paradigms were chemokines such as Ccl2 and Ccl7, suggesting an important role for neuroimmune modulation. Collectively these data will aid in defining pro-regenerative and pro-degenerative mechanisms in motor neurons.
Cancer immunology research
Reschke, R;Shapiro, JW;Yu, J;Rouhani, SJ;Olson, DJ;Zha, Y;Gajewski, TF;
PMID: 35977003 | DOI: 10.1158/2326-6066.CIR-22-0362
Immune checkpoint blockade is therapeutically successful for many patients across multiple cancer types. However, immune-related adverse events (irAE) frequently occur and can sometimes be life threatening. It is critical to understand the immunologic mechanisms of irAEs with the goal of finding novel treatment targets. Herein, we report our analysis of tissues from patients with irAE dermatitis using multiparameter immunofluorescence (IF), spatial transcriptomics, and RNA in situ hybridization (RISH). Skin psoriasis cases were studied as a comparison, as a known Th17-driven disease, and colitis was investigated as a comparison. IF analysis revealed that CD4+ and CD8+ tissue-resident memory T (TRM) cells were preferentially expanded in the inflamed portion of skin in cutaneous irAEs compared with healthy skin controls. Spatial transcriptomics allowed us to focus on areas containing TRM cells to discern functional phenotype and revealed expression of Th1-associated genes in irAEs, compared with Th17-asociated genes in psoriasis. Expression of PD-1, CTLA-4, LAG-3, and other inhibitory receptors was observed in irAE cases. RISH technology combined with IF confirmed expression of IFNγ, CXCL9, CXCL10, and TNFα in irAE dermatitis, as well as IFNγ within TRM cells specifically. The Th1-skewed phenotype was confirmed in irAE colitis cases compared with healthy colon.
Mucosal IFNγ production and potential role in protection in Escherichia coli O157:H7 vaccinated and challenged cattle
Schaut, RG;Palmer, MV;Boggiatto, PM;Kudva, IT;Loving, CL;Sharma, VK;
PMID: 33963240 | DOI: 10.1038/s41598-021-89113-7
Shiga-toxin producing Escherichia coli O157:H7 (O157)-based vaccines can provide a potential intervention strategy to limit foodborne zoonotic transmission of O157. While the peripheral antibody response to O157 vaccination has been characterized, O157-specific cellular immunity at the rectoanal junction (RAJ), a preferred site for O157 colonization, remains poorly described. Vaccine induced mucosal O157-specific antibodies likely provide some protection, cellular immune responses at the RAJ may also play a role in protection. Distinct lymphoid follicles were increased in the RAJ of vaccinated/challenged animals. Additionally, increased numbers of interferon (IFN)γ-producing cells and γδ + T cells were detected in the follicular region of the RAJ of vaccinated/challenged animals. Likewise, adjuvanted-vaccine formulation is critical in immunogenicity of the O157 parenteral vaccine. Local T cell produced IFNγ may impact epithelial cells, subsequently limiting O157 adherence, which was demonstrated using in vitro attachment assays with bovine epithelial cells. Thus, distinct immune changes induced at the mucosa of vaccinated and challenged animals provide insight of mechanisms associated with limiting O157 fecal shedding. Enhancing mucosal immunity may be critical in the further development of efficacious vaccines for controlling O157 in ruminants and thus limiting O157 transmission to humans.
Influence of the microenvironment on modulation of the host response by typhoid toxin
Martin, OCB;Bergonzini, A;Lopez Chiloeches, M;Paparouna, E;Butter, D;Theodorou, SDP;Haykal, MM;Boutet-Robinet, E;Tebaldi, T;Wakeham, A;Rhen, M;Gorgoulis, VG;Mak, T;Pateras, IS;Frisan, T;
PMID: 33826883 | DOI: 10.1016/j.celrep.2021.108931
Bacterial genotoxins cause DNA damage in eukaryotic cells, resulting in activation of the DNA damage response (DDR) in vitro. These toxins are produced by Gram-negative bacteria, enriched in the microbiota of inflammatory bowel disease (IBD) and colorectal cancer (CRC) patients. However, their role in infection remains poorly characterized. We address the role of typhoid toxin in modulation of the host-microbial interaction in health and disease. Infection with a genotoxigenic Salmonella protects mice from intestinal inflammation. We show that the presence of an active genotoxin promotes DNA fragmentation and senescence in vivo, which is uncoupled from an inflammatory response and unexpectedly associated with induction of an anti-inflammatory environment. The anti-inflammatory response is lost when infection occurs in mice with acute colitis. These data highlight a complex context-dependent crosstalk between bacterial-genotoxin-induced DDR and the host immune response, underlining an unexpected role for bacterial genotoxins.
Strasser K, Birnleitner H, Beer A, Pils D, Gerner MC, Schmetterer KG, Bachleitner-Hofmann T, Stift A, Bergmann M, Oehler R.
PMID: - | DOI: 10.1080/2162402X.2018.1537693
T cells in colorectal cancer (CRC) are associated with improved survival. However, checkpoint immunotherapies antagonizing the suppression of these cells are ineffective in the great majority of patients. To better understand the immune cell regulation in CRC, we compared tumor-associated T lymphocytes and macrophages to the immune cell infiltrate of normal mucosa. Human colorectal tumor specimen and tumor-distant normal mucosa tissues of the same patients were collected. Phenotypes and functionality of tissue-derived T cells and macrophages were characterized using immunohistochemistry, RNA in situ hybridization, and multiparameter flow cytometry. CRC contained significantly higher numbers of potentially immunosuppressive CD39 and Helios-expressing regulatory T cells in comparison to normal mucosa. Surprisingly, we found a concomitant increase of pro-inflammatory IFNγ -producing T cells. PD-L1+ stromal cells were decreased in the tumor tissue. Macrophages in the tumor compared to tumor-distant normal tissue appear to have an altered phenotype, identified by HLA-DR, CD14, CX3CR1, and CD64, and tolerogenic CD206+macrophages are quantitatively reduced. The prognostic effect of these observed differences between distant mucosa and tumor tissue on the overall survival was examined using gene expression data of 298 CRC patients. The combined gene expression of increased FOXP3, IFNγ, CD14, and decreased CD206 correlated with a poor prognosis in CRC patients. These data reveal that the CRC microenvironment promotes the coexistence of seemingly antagonistic suppressive and pro-inflammatory immune responses and might provide an explanation why a blockade of the PD1/PD-L1 axis is ineffective in CRC. This should be taken into account when designing novel treatment strategies.
Inflamm Bowel Dis. 2017 Nov;23(11):1950-1961.
Shouval DS, Konnikova L, Griffith AE, Wall SM, Biswas A, Werner L, Nunberg M, Kammermeier J, Goettel JA, Anand R, Chen H, Weiss B, Li J, Loizides A, Yerushalmi B, Yanagi T, Beier R, Conklin LS, Ebens CL, Santos FGMS, Sherlock M, Goldsmith JD, Kotlarz D, Glover SC, Shah N, Bousvaros A, Uhlig HH, Muise AM, Klein C, Snapper SB.
PMID: 29023267 | DOI: 10.1097/MIB.0000000000001270
Abstract BACKGROUND: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4 T-cell function. METHODS: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4 T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. RESULTS: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4 T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1β leads to enhanced production of IL17A. CONCLUSIONS: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.
Boxberg M, Leising L, Steiger K, Jesinghaus M, Alkhamas A, Mielke M, Pfarr N, Götz C, Wolff KD, Weichert W, Kolk A.
PMID: 30530592 | DOI: 10.4049/jimmunol.1800242
Immunotherapy shows promising results and revolutionizes treatment of oral squamous cell carcinoma (OSCC). The immunologic microenvironment might have prognostic/predictive implications. Morphologic immunologic parameters (inflammatory infiltrate, stromal content, and budding activity [BA] [potentially indicating epithelial–mesenchymal transition]) were evaluated in 66 human primary therapy-naive OSCCs. Intraepithelial/stromal tumor-infiltrating lymphocytes (TILs; CD3+/CD4+/CD8+/CD4+FOXP3+/IL-17A+) were quantified, and ratios were calculated. HLA class I in tumor cells was evaluated immunohistochemically. mRNA in situ hybridization to detect IFN-γ was performed. Analysis was performed within invasive front (IF) and tumor center (TCe). Decreased HLA expression was associated with low TIL density, pronounced stromal content, and high BA; IFN-γ in TILs was correlated with high-density TILs; and IFN-γ in tumor cells was correlated with absence of BA (p < 0.05). Heterogeneity of parameters (TCe/IF) was rare. Low density of stromal CD4+FOXP3+ TILs within TCe and IF was identified as an independent prognostic factor for poor overall, disease-specific, and disease-free survival (p ≤ 0.011). Refining prognostication in OSCC with high-density CD4+FOXP3+ infiltrate within TCe and/or IF, high FOXP3:CD4 ratio was significantly correlated with favorable outcome in this subgroup. Furthermore, high-stromal CD8:CD4 ratio was found to be an independent favorable prognostic factor. In summary, immunologic parameters were closely intertwined. Morphologic correlates of epithelial–mesenchymal transition were associated with downregulation of HLA and decreased inflammation. Heterogeneity was infrequent. Low-density stromal CD4+FOXP3+ infiltrate within TCe and IF was an independent poor prognostic factor. Stratification of cases with high-density CD4+FOXP3+ TILs by FOXP3:CD4 ratio enables refinement of prognostication of this subgroup. CD8:CD4 ratio was identified as an independent prognostic factor.
Sasaki, K;Hayamizu, Y;Murakami, R;Toi, M;Iwai, K;
PMID: 37060248 | DOI: 10.1002/1873-3468.14623
Tumor-elicited inflammation confers tumorigenic properties, including cell death resistance, proliferation, or immune evasion. To focus on inflammatory signaling in tumors, we investigated linear ubiquitination, which enhances the nuclear factor-κB signaling pathway and prevents extrinsic programmed cell death under inflammatory environments. Here, we showed that linear ubiquitination was augmented especially in tumor cells around a necrotic core. Linear ubiquitination allowed melanomas to tolerate the hostile tumor microenvironment and to extend a necrosis-containing morphology. Loss of linear ubiquitination resulted in few necrotic lesions and growth regression, further leading to repression of innate anti-PD-1 therapy resistance signatures in melanoma as well as activation of interferon responses and antigen presentation that promote immune-mediated tumor eradication. Collectively, linear ubiquitination promotes tumor-specific tissue remodeling and the ensuing immune evasion.
